BIOSYNTHESIS OF ADENOVIRUS EARLY RNAS

癌细胞的异常特性部分是由于 一些转录因子（TF）的不适当激活， 其他人的失活。了解TF的功能应该允许 设计治疗方法来改变导致糖尿病的异常TF 肿瘤发生大多数调节性转录因子是具有不同DNA的模块蛋白 结合和激活结构域。DNA结合的机制 结构域的功能已经很好地理解，但对如何理解却知之甚少。 激活域功能。激活结构域刺激pol II 从一个复杂的预引发复合体引发， TF和pol II。我们建议研究的激活域的 强病毒活化剂腺病毒2 E1 A、爱泼斯坦-巴尔病毒Zta和 单纯疱疹病毒VP 16;以及重要的肿瘤抑制因子 第53页。这些研究依赖于纯化功能性TFIID的能力， 由TATA结合蛋白组成的复杂通用转录因子 启动前起始复合物装配的蛋白质（TBP）和TAF 在带有TATA框的促销员处。一种次要的核蛋白CR 3BP， 与E1 A辅活化剂的预测性质相一致：它 结合野生型E1 A激活结构域，但不结合点突变缺陷型 在活化过程中结合TBP。如果额外的实验 与E1 A共激活因子功能一致，编码CR 3BP的cDNA将 克隆并用于分析其转录活性。上的区域 与E1 A和其他活化结构域相互作用的TBP表面， 一般TF和TAF将通过引入氨基酸 取代到其91个表面氨基酸残基中的每一个， 不要接触DNA含有结合一般TF的突变TBP的TFIID和 TAF通常但有缺陷的激活转录将被激活。 分离并用于活化结构域结合的测定， preinitiation复杂的组装，以确定哪一步在激活 组装、pol II启动或启动子清除有缺陷。ZTA 激活启动子DNA上TFIID和TFIIA的组装，但这 刺激不足以完全解释Zta激活。 在D-A组装之后的预引发复合物组装中的步骤将 使用能够分辨DNA蛋白质的琼脂糖凝胶进行测定 >10（6）Da的复合物和凝胶过滤测定以检测因子 与质粒模板结合。pol II启动的激活和 还将分析启动子清除。类似的研究将分析 通过E1 A和p53的激活以及通过激活剂的组合的激活 在合成的模板上有两种激活剂的结合位点。 针对最近克隆的pol亚基的特异性抗体 第三部分将利用TFIIIC因子分析TFIIIC的作用机制 调节响应病毒感染和生长因子。

The abnormal properties of cancer cells are due in part to the inappropriate activation of some transcription factors (TFs) and the inactivation of others. Understanding how TFs function should allow the design of therapies that modify the abnormal TFs that contribute to oncogenesis. Most regulatory TFs are modular proteins with distinct DNA binding and activation domains. The mechanisms by which DNA binding domains function are well understood, but little is known about how activation domains function. Activation domains stimulate pol Il initiation from a complicated preinitiation complex composed of general TFs and pol Il. We propose to study the activation domains of the strong viral activators adenovirus 2 E1A, Epstein-Barr Virus Zta, and herpes simplex virus VP16; as well as the important tumor suppressor p53. The studies depend on the ability to purify functional TFIID, the complex general transcription factor composed of the TATA-binding protein (TBP) and TAFs that initiates preinitiation complex assembly at promoters with a TATA-box. A minor nuclear protein, CR3BP, has been identified with the predicted properties of an E1A coactivator: it binds the wt E1A activation domain, but not to point mutants defective in activation that do bind TBP. If additional experimentation is consistent with E1A coactivator function, a cDNA encoding CR3BP will be cloned and used to analyze its transcriptional activity. Regions on the surface of TBP that interact with E1A and other activation domains, general TFs and TAFs will be analyzed by introducing amino acid substitutions into each of its 91 surface amino acid residues that do not contact DNA. TFIID containing mutant TBPs that bind general TFs and TAFs normally but are defective for activated transcription will be isolated and used in assays of activation domain binding and preinitiation complex assembly to determine which step in activated assembly, pol II initiation or promoter clearance is defective. Zta activates assembly of TFIID and TFIIA on promoter DNA, but this stimulation is not sufficient to account completely for Zta activation. Steps in preinitiation complex assembly subsequent to D-A assembly will be assayed using agarose gels capable of resolving DNA protein complexes of >10(6) Da and a gel filtration assay to detect factor binding to plasmid templates. Activation of pol II initiation and promoter clearance will also be analyzed. Similar studies will analyze activation by E1A and p53 and activation by combinations of activators on synthetic templates with binding sites for two types of activators. Specific antibodies raised against a recently cloned subunit of the pol III factor TFIIIC will be used to analyze the mechanism of TFIIIC regulation in response to viral infection and growth factors.